The following includes information that may be useful in understanding the present invention. It is not an admission that any of the information, publications or documents specifically or implicitly referred to or referenced herein is prior art, or essential, to the presently described or claimed inventions. All publications and patents mentioned in this specification are incorporated herein by reference in their entirety.
The advent of highly sensitive (hs) assays for the detection of cardiac troponins (cTn) has significantly altered our understanding of troponin in the circulation and altered aspects of its use for the diagnosis of acute myocardial infarction (MI; Thygesen et al. (2012) Circulation 126:2020-2035; Keller et al. (2009) N. Eng. J. Med. 361:868-877; Reichlin et al. (2009) N. Eng. J. Med. 361:858-867). These assays have resulted in major improvements in time to diagnosis of myocardial infarction (MI), but this has come at the cost of increasing numbers of patients who have elevated levels of troponin above 99th percentile guidelines, but do not in fact have MI (Reichlin et al. (2012) Am. J. Med. 125:1205-1213; Thygesen et al. (2012) Eur. Heart J. 33:2252-2257; Hammerer-Lercher et al. (2013) J. Am. Heart Assoc. 2(3):e000204.doi:10.1161/JAHA.113.000204). This imbues significant reductions in assay specificity and as a consequence several biomarker based strategies have been suggested to help hscTn measurement (Eggers et al, (2008) Eur. Heart J. 29:2327-2335; Haaf et al. (2011) Am. J. Med. 124:731-739; Maisel et al. (2013) J. Am. Coll. Cardiol. 62:150-160; Cullen et al. (2013) J. Am. Coll. Cardiol. 62:1242-1249). One potential strategy to overcome these limitations is consideration of total potential cardiac troponin produced, including upstream open reading frame peptides.
Upstream open reading frames (uORFs) are amino acid coding sequences defined by a start and stop codons upstream (5′) of the main coding region in proteins. Approximately 40-50% of the human and mouse transcriptome contain uORFs (Calvo et al. (2009) Proc. Natl. Acad. Sci. 106:7507-7512; Iacono et al. (2005) Gene 349:97-105) and they are less frequent than expected by chance (Neafsey et al. (2007) Mol. Biol. Evol. 24:1744-1751) suggesting they are under selective pressure. However, uORFs are overrepresented in the protein subgroup containing transcription factors, growth factors, proto-oncogenes and their receptors (Davuluri et al. (2000) Genome Res. 10:1807-1816). Adding to their complexity, uORFs are extremely diverse varying in position in relation to the cap and main ORF AUG, number per transcript and length (Calvo et al. (2009) Proc. Natl. Acad. Sci. 106:7507-7512; Somers et al, (2013) Int. J. Biochem. Cell Biol. 45:1690-1700). In mammalian cells there is limited experimental evidence concerning direct translational actions of uORFs upon cellular function and there is no evidence that uORF-derived peptides are present in the circulation. A single report has documented that the glucocorticoid receptor transcript 1A (GR-1A) possesses 5 uORF start sites in its 1026 bp 5′-UTR. One of these uORFs encodes a 93 amino acid peptide that was detected in the nucleus, cytosol and plasma membrane of mouse and human cell cultures and was found to play a role in promoting GR-1A translation (Diba et al. (2001) J. Cell. Biochem. 81:149-161).
The Applicants demonstrate here that uORF peptides are present in the 5′-UTR of the human cardiac troponin T (TnT) gene and surprisingly provide the first direct evidence to demonstrate that a cardiac TnTuORF peptide is indeed present in the human circulation and that its assay quantitation has utility in the prognosis and diagnosis of cardiac disorders, and in improving the sensitivity and false positive performance of established biomarkers for cardiac disease, including myocardial infarction.